Undifferentiated pleomorphic sarcoma (UPS) is a soft tissue sarcoma, with one of the worst prognosis. We will build on our discovery that UPS contains a small subpopulation of tumor initiating cells (TICs) that are enhanced for the ability t form tumors when transplanted into immune-deficient mice. Our proof of principle data showed that therapeutically targeting signaling pathways activated in the TIC population inhibits growth in UPS tumors established as xenografts in mice. In our preliminary data, we identified the chondroitin sulfate proteoglycan, NG2, as expressed in the TIC subpopulation in human and mouse UPS tumors. Our hypothesis is that the TIC fraction in UPS is responsible for maintaining UPS self-renewal and tumor growth, and that therapeutically targeting the TIC fraction can be developed into an effective UPS treatment. We will test this hypothesis by answering the following questions: 1) Will the eradication of TICs cause the degeneration of UPS? We found that deletion of Ng2 expressing cells in mouse UPS tumors, by driving Diphtheria Toxin in tumors established as allografts, will cause degeneration of tumors. Using flippase to activate oncogenic alleles to drive UPS development in mice, and cre-recombinase to drive Diphtheria Toxin in Ng2 expressing, we will be able to delete the TIC population in tumors in-situ, and determine if this causes degeneration of UPS tumors, prevents recurrence, or inhibits metastatic capacity. 2) Are individual clones within the Ng2 expressing population stable over time and resistant to conventional therapies? Ng2 expressing cells could be composed of cells that survive long term in tumors, giving rise to daughter cells that maintain the tumor over time; or they may represent different cells, each having TIC characteristics at different points in time. To determine which is the case, we will use the Confetti allele to label Ng2 expressing cells in mouse UPS tumors with different colored fluorescent proteins labeling individual clones. How these cell populations behave as the tumors grow, with serial transplantation, and after treatment using conventional therapies will determine which one of these possibilities is the case. 3) Can targeting NG2 be used to treat UPS? To determine how targeting Ng2 expression regulates tumor growth and self-renewal, NG2 will be knocked-down with shRNA in human UPS tumors established as xenografts in immunodeficient mice and NG2 will be deleted in primary mouse UPS tumors initiated by Cre recombinase that drives expression of oncogenes and recombines conditional Ng2 null alleles. Immunotherapy to NG2 will be examined in a similar manner in UPS tumors established as xenografts in immunodeficient mice. In addition to proving important biologic insight into sarcomas, these data will determine if short or long term therapeutic targeting of the TIC population will be more effective in UPS treatment, and inform a novel approach to develop a therapy based on targeting the UPS TIC.